Biochemical properties of oncornavirus polypeptides

Biochimica et Biophysica Acta 355 (1974) 220-235 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 87009

BIOCHEMICAL

PROPERTIES OF ONCORNAVIRUS

POLYPEPTIDES

DANI P. BOLOGNESI", GUDRUN HUPER", ROBERT W. GREEN" and THOMAS GRAF b a Department of Surgery, Duke University Medical Center, Durham, N.C. 27710 (U.S.A.) attd Max-Planck-lnstitut fiir Virusforschung, Tiibingen (G.F.R.) (Received April 8th, 1974)

CONTENTS 1. 11. Ill. IV.

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General properties of oncornavirus polypeptides . . . . . . . . . . . . . . . . . Separation of the major structural proteins of oncornavirus polypeptides by gel filtration in guanidine hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Charge properties of oncornavirus polypeptides . . . . . . . . . . . . . . . . . . VI. Virus structural components containing carbohydrate . . . . . . . . . . . . . . . VII. Polypeptide content of avian oncornaviruses of different subgroups . . . . . . . . . VIII. Comparative properties of avian and mammalian oncornavirus polypeptides . . . . . IX. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

220 220 221 222 225 227 229 231 233 233 234

I. SUMMARY The m a j o r structural polypeptides o f avian and m a m m a l i a n o n c o r n a v i r u s e s display a variety o f unique physical a n d chemical properties,

T e c h n i q u e s were

designed to emphasize certain qualities o f the individual proteins and glycoproteins which facilitate their identification in c o m p l e x mixtures.

II. I N T R O D U C T I O N Progress during the last several years has resulted in the el u ci d at i o n o f the m e c h a n i s m o f replication o f a variety o f bacterial and animal viruses. Th e sequence o f events leading to the f o r m a t i o n o f c o m p l e t e virus particles has been described in

Abbreviations: AMV, Avian myeloblastosis virus; MuLV (Friend), Murine leukemia virus (Friend strain); FeLV (Rickard), Feline leukemia virus (Rickard strain); GuHC1, Guanidine hydrochloride; SR-RSV-B, Schmidt-Ruppin strain of Rous sarcoma virus (RSV) of subgroup B; PR-RSV, Prague strain of RSV; B-77, Bratislava strain of RSV; RAV, Rous associated virus.

221 considerable detail through analyses of virus gene products at various stages during the infectious cycle. Most of the early advances were made by studying the intracellular localization and synthesis of the structural components of the virions. In the case of RNA tumor viruses, very little is known about the mechanism of replication of these agents. Although it has been well established that the particles mature at the cell surface, it is not understood how and where the individual nucleic acid and protein components are synthesized. Progress in this area is hampered by the very small percentage of virus-specific nucleic acid and protein syntheses in comparison to those of the host ceil. Probably more troublesome, however, is that the RNA tumor virus systems, unlike other RNA virus systems, do not lend themselves to the use of metabolic inhibitors which permit differentiation of host functions from those of the replicating virus. Recently, a great deal of information has been obtained about the various structural components of the virus particles, including the virus proteins, nucleic acids, and enzymes. Progress in this area indicates that the virus RNA as well as the reverse transcriptase can be implemented directly, or utilized to prepare reagents applicable to the analysis of cells for the presence of virus genetic material. Extremely sensitive techniques have been devised to determine the number of copies of the virus genome contained in the DNA of a variety of cell types [1,2], as well as the extent of intracellular transcription of the virus DNA into virus RNA [3]. In contrast, relatively little progress has been made concerning the translation of virus genetic material into protein products. Since virus gene products undoubtedly play important roles in processes related to infection, transformation, and tumor formation, it is important to study their properties in detail.

IlI. GENERAL PROPERTIES OF ONCORNAVIRUS POLYPEPTIDES Studies in this and other laboratories have indicated that both avian and mammalian oncornaviruses are characterized by a rather complex polypeptide composition. Analyses by sodium dodecylsulfate gel electrophoresis resolved at least 18 distinct polypeptides in avian myeloblastosis virus [4] and as many as 26 in Rous sarcoma virus [5]. Patterns of similar complexity can be discerned also in the mammalian oncornaviruses studied thus far [6-9]. Although acrylamide electrophoresis in the presence of sodium dodecylsulfate separates most proteins with high resolution on the basis of molecular weight, there are many polypeptides which behave anomalously in this system [10]. Furthermore, the molecular weight of glycoproteins cannot be estimated accurately on the basis of migration in these gels [11 ]. Gel filtration in guanidine hydrochloride (GuHCI) has also been used to separate oncornavirus polypeptides [12,13]. This technique, like sodium dodecylsulfate acrylamide gel electrophoresis, separates protein chains on the basis of molecular weight and is particularly effective, in contrast to the former, for low molecular weight proteins [14-16]. For avian oncornaviruses, an additional component was

222 resolved which co-electrophoresed with the most rapidly migrating polypeptide in sodium dodecylsulfate gel electrophoresis [12]. This method is not as useful f o r p r o teins with molecular weights greater than 40 000, and has not been adapted for rapid analytical purposes. Techniques utilizing charge differences a m o n g avian oncornavirus polypeptides have also been employed to separate the various components [17,18]. Iso-electric focusing has been used extensively for purification of the major polypeptide of m a m malian oncornaviruses [6,7] but sparingly in avian virus work [19]. Like the guanidine column, this procedure is usually included for large scale isolation rather than for analytical purposes. Studies performed thus far show that no single technique is suitable for complete separation of all the proteins and glycoproteins of avian or mammalian oncornaviruses. Correct identification of the structural proteins can only be achieved by multiple determination in various analytical systems. This communication is an attempt to illustrate the applicability of the more useful techniques and to describe some of the properties of the major structural polypeptides which facilitate their identification.

IV. SEPARATION OF THE MAJOR STRUCTURAL PROTEINS OF ONCORNAV1RUSES BY GEL FILTRATION IN GuHCI The information presented in Fig. 1 is similar to that reported by Fleissner [12] for avian viruses and Nowinski et al. [13 ] for mammalian agents and is re-introduced here for direct comparative purposes. The proteins are designated on the basis of molecular weight in G u H C I (Table I) using a recently adopted nomenclature [30].

TABLE I DESIGNATION OF ONCORNAVIRUS POLYPEPTIDES Proteins (p) and glycoproteins (gp) will be designated on the basis of molecular weight. Values below 30 000 are assigned on the basis of elution volume from GuHCI-Sepharose columns. Values greater than 31 000 are derived from migration in sodium dodecylsulfate acrylamide gels. Molecular weight values will be times l0 -a (e.g. p31 = p31 000).* Avian

Murine

Feline

pl0 p12 p15 p19 p27 gp37 gp85

pl0 p12 p15 w p31 gp45 gp77

pl0 p12 p15 __ p31 -gp72

* Agreed upon at a colloquium held at the SIoan-Kettering Institute for Cancer Research, June 4-5, 1974 (August et al. [30J).

223 1.0

0.8 E ,=

o

06

04

~a

02 27,000

120

140

[ 180

160

1 200

19,000 15,000 I

220 240 260 Fraction Number

280

300

320

340

360

,61 ~4

ol2

c-a

";10

08

~o6 04 02 ~ _J 120

i

160

200

31,000 240

~5,000~ I

l

l

I

280

320

360

400

Froclion Number

18 1,6

14 12 I0 ~0,8 06: 0, 30,000

02 120

160

200

15,000

240

280

320

Fr0cti0a Number

Fig. I. Gel filtration of oncornavirus polypeptides in G u H C I . Amounts equivalent to 50 mg of virus protein were disrupted with GuHC1 and separated by gel filtration using Sepharose 6B in the presence of 6 M G u H C I [28]. Absorbance (280 nm) was read continuously in a Beckman DU Spectrophotometer (Beckman Instruments, lnc.) equipped with a flow c~ll. (A) AMV; (B) Murine Leukemia virus (MuLV) (Friend); (C) FeLV (Rickard). Elution is from left to right.

224

27

---7 L~..,.o.,~

Fig. 2. Analysis of AMV GuHCI fractions by sodium dodecylsulfate gel electrophoresis Following renaturation from GuHCI, proteins were treated with 0.1 M dithiothreitol and 0.1% sodium dodecylsulfate and electrophoresed in the presence of sodium dodecylsulfate as described previously [26]. From left to right AMV, gp85, p27, p19, p15, p12 and pt0. Migration is from top to bottom and staining is with Coomassie blue.

One can see that the effectiveness of the column resides primarily in the molecular weight range between l0 000 and 31 000. Changing the resin would improve the resolution in the higher molecular weight range, but would concomitantly result in a poorer resolution of the lower molecular weight components [28]. When the top fractions of the major peaks from the AMV column were subjected to electrophoresis in sodium dodecylsulfate acrylamide gels, we obtained the results illustrated in Fig. 2. One can see that the column effected an excellent separation of the major proteins (p10-p27), but not of the higher molecular weight materials (gp85). As pointed out originally by Fleissner [l 2], pl 0 and p 15 co-electrophorese in the sodium dodecylsulfate gel and that pl2 migrates slower than pl5 in spite of its lower molecular weight in GuHCI. A similar situation exists for the separation of MuLV (Friend) structural components except that the order of elution from the column and migration in the sodium dodecylsulfate gel is the same (see Green et al. (31)). This is not the case for all mammalian agents since the FeLV (Rickard) polypeptides pl0 and pl2 which are well separated on the GuHCl column co-migrate in the sodium dodecylsulffate gel (Fig. 3). The variation in other mammalian oncornaviruses such

225

Avian

Murine

gp85

Feline

gp 77

qp72

37 p31 p31

~pfo, pf5 p12

p15 p12

p15

plO

plO, p12

Fig. 3. Polypeptide patternsofAMV, MuLV(Friend) and FeLV (Rickard)insodiumdodecylsulfate gel electrophoresis. Virus preparations containing about 1 mg/ml protein were treated with acetone (see Fig. 6) and electrophoresed as described above. From left to right: AMV, MuLV (Friend), FeLV (Rickard).

as the simian sarcoma virus (SSV-1) is even more pronounced (unpublished), but more needs to be known about these proteins before it is possible to compare them directly to those of MuLV and FeLV.

v. CHARGE PROPERTIES OF ONCORNAVIRUS POLYPEPTIDES In view of the anomalies in the sodium dodecylsulfate system for the avian and feline proteins, other procedures to analytically separate the polypeptides were examined. Electrophoresis in 6 M urea at low p H (3.2)was found to be a method which provides useful information. As shown in Fig. 4, the major components can

226

l.o oB -

5,

plO

Pi I

p27 p19 pjl

~o

6

p15,p12 p12

p

3

6

9

3

6

Cenlimelers

Fig. 4. Electrophoresis of oncornavirus polypeptides in acrylamide gels containing 6 M urea at pH 3.2. Virus preparations were treated with 6 M urea in the presence of 0.1 M dithiothreitol and l M acetic acid for 60 rain at 37 °C. Preparation of 7.5 ~ acrytamide gels in the presence of 6 M urea and electrophoresis of the samples were as described by Panyim and Chalkley [29]. Polypeptide patterns were obtained by scanning in a Gilford Spectrophotometer at 530 nm. (A) AMV; (B) MuLV (Friend). Electrophoresis is from left to right and from anode ( ÷ ) to cathode (--).

be separated quite well and their identity established by electrophoresis of the purified proteins (from GuHC1). On the basis of their rapid migration properties, one can see that avian myeloblastosis virus (MV) pl2, MuLV pl0, and FeLV pl0 (not shown) must be strongly basic proteins and this was confirmed by amino acid analyses (in preparation). Under these conditions, the major glycoprotein of A MV (gp85) does not penetrate the gel suggesting that it is strongly acidic. Very few of the minor high molecular weight bands visible in the sodium dodecylsulfate gels are discernible in this system. Either they did not penetrate the gel or they co-migrated with the other bands. It is curious that pl0 and pl5 migrate to the same position in this system just as they do in the sodium dodecylsulfate gels. If the pH is raised to 7.2 and the polarity is reversed, a rather interesting situation occurs. Whereas most of the virion protein components remain unresolved toward the top of the gel, AMV pl0 and MuLV pl2 are clearly separated under these conditions (Fig. 5). This suggests that like the glycoproteins, these are acidic components. They also behave like acidic proteins on DEAE columns and their amino acid composition reflects this (in preparation). At pH 3.2, however, these components result as multiple bands on the gel revealing some heterogeneity.

227

pi2 ~

iiii,~~

::

Fig. 5. Electrophoresis of AMV polypeptides in urea-containing gels at pH 7.2. The gel system is similar to that above except that the electrophoresis buffer was a Tris buffer at pH 7.2 [26] and a 5 ~o acrylamide gel was used. Left, AMV, pH 3.2; middle, AMV, pH 7.2; right, pl0, pH 7.2. Electrophoresis is from top (--) to bottom (÷).

v1. VIRUS STRUCTURAL COMPONENTS CONTAINING CARBOHYDRATE Previous studies have shown that avian oncornaviruses contain two glycoproteins, as part of their outer surface structure, which represent the type specific antigens of the virus [27,32]. The major glycoprotein (gp85) contains a considerable amount of carbohydrate (about 20 ~ by weight) most of which is glucosamine (in preparation). The minor glycoprotein (gp37) contains much less carbohydrate and and is rather difficult to detect by specific labeling [32] or staining [27] of the carbohydrate. A similar situation exists for mammalian oncornaviruses which also reveal a major glycoprotein [8,33] with type specific properties [9,34] as well as hemagglutinating capacity [35,36]. G r o u p [9,33,34] and interspecies [33,34] antigenic specificities have also been detected in this component. The identity of a minor glycoprotein in

228

gp85

Fig. 6. Components containing carbohydrate and the effect of acetone. Virus preparations were treated with 10 volumes of acetone tor 5 min at room temperature. The control was untreated. Following centrifugation at 2500 x g for 10 min, the sedimentable material was analyzed by sodium dodecylsulfate gel electrophoresis as described in Fig. 2. From left to right: AMV ~ acetone (Coomassie blue); AMV ~ acetone (periodic acid Schiff); AMV (Coomassie blue); AMV (periodic acid Schiff).

mammalian agents corresponding to avian gp37 has not been clearly established. In the case of AMV, the two glycoproteins make up about 60 ~ of the glucosamine incorporated by the virus and can be stained with periodic acid Schiffreagent. The remainder of the labeled material which, like the glycoproteins, is stained with periodic Shift can be found in a diffuse material in the low molecular weight region of the sodium dodecylsulfate gel (Fig. 6). This component interferes somewhat with the migration of pl0, p15 and p12. Recent studies have shown that it represents glycolipid [37] and can be removed by precipitation of the virus through acetone or ethanol (Fig.' 6). It presumably represents a portion of the cellular membrane acquired by the virus during maturation [37]. Two mammalian agents studied, M u L V and FeLV, likewise possess a similar acetone removable periodic Schiffstaining component. We have observed that prior treatment with acetone in this way renders the polypeptide preparations of all the viruses studied much more amenable to both sodium dodecylsulfate gel electrophoresis (the bands are sharper and more discrete) and gel

229 SR-RSV-A -B p27

A

SR-RSV-B C.

PR-RSV- C

OE

p~O,,pl5--

O~ E

i

o~ I

D

B-77-C

E

F

RSV (RAV-O)-E

O~

O~

30

60

0

30 60 Cenhmefers Gel

30

60

90

Fig. 7. Analysis of avian sarcoma virus polypeptides by sodium dodecylsulfate gel electrophoresis. Purified virus preparations were treated with acetone and prepared for electrophoresis in the presence ot sodium dodecylsulfate. The origin of strains used has been described [21-24]. They were propagated on chick embryo ceils known to be free of endogenous virus activity [25]. Purification of the agents was according to previously established procedures [26,27]. After staining witb Coomassie blue, the polypeptide patterns were analyzed by scanning in a spectrophotometer at 530 nm as (A) SR-RSV-A; (B) SR-RSV-B; (C) PR-RSV-C; (D) B-77-C; (E) SR-RSV-D; (F) RSV(RAV-O)-E. Migration is from left to right.

filtration in G u H C l . Repeated precipitations t h r o u g h acetone, however, result in some solubilization o f the basic proteins, avian p l 2 and m a m m a l i a n pl0.

vii. POLYPEPTIDE CONTENT OF AVIAN ONCORNAVIRUSES OF DIFFERENT SUBGROUPS A series of avian oncornaviruses was investigated to determine if there were any strain specific differences in the quantitative or qualitative aspects of the major polypeptides which are well separated. With the exception o f Rous sarcoma virus (RSV) (Rous associated virus, RAV-O), only non-defective viruses were used in this study and all were clone purified. As shown in Fig. 7, representative agents of the five subgroups revealed a polypeptide pattern which was grossly similar following sodium dodecylsulfate gel electrophoresis. Some variation in the p r o p o r t i o n of the various polypeptides

230 A

p 7 SR-RSV-A

B

SR-RSV-B

E

PR-RSV-C

08 plO,plS

04

[

gi

.

.

.

.

.

~ 9

3 o~

0

30

60

90 0

3(9

60

90 0

30

60

90

Cenhmeters Gel

Fig. 8. Analysis of avian sarcoma virus polypeptides by urea gel electrophoresis at pH 3.2. Purified virus preparations were disrupted with urea, electrophoresed as described above, and analyzed by scanning spectrophotometry. (A) SR-RSV-A; (B) SR-RSV-B; (C) PR-RSV-C; (D) B-77-C; (E) SR-RSV-D; (F) RSV(RAV-O)-E. Migration is from left ( ÷ ) to right (--).

exists among all of the agents. Only in the case of the subgroup C agents, however, were notable differences observed. The two agents tested (Prague strain of RSV (PR-RSV-C) and Bratislava strain of RSV (B-77)) both contain a much lesser quantity of the pl 9 polypeptide. Examination of the same agents using electrophoresis in the presence of urea of pH 3.2 yielded the patterns illustrated in Fig. 8. The rate of migration of most of the respective polypeptides is similar among the viruses with the exception of p19. One can see that p19 of Schmidt-Ruppin strain of RSV of subgroup B (SR-RSV-B) (arrow) migrates more rapidly than p19 of SR-RSV-A. The corresponding polypeptide of PR-RSV-C is hardly discernible and may be co-migrating with one of the other polypeptides. In contrast, that of another subgroup C virus, B-77 is clearly distinguishable. A slight variability in migration of the basic protein, pl2, is also evident. These results suggest that among avian sarcoma viruses, most of the major polypeptides are present in roughly the same proportions and have similar molecular weights and charge properties. One notable exception is pl9. This polypeptide is present in significantly lower amounts in subgroup C viruses and exhibits a variability in rate of migration in urea-containing gels among some of the viruses studied. Recent work employing peptide mapping of p19 from two distinct strains, revealed differences in peptide content [54]; and immunological studies employing quantitative radioimmunoassay indicated the presence of distinguishable antigenic determinants [55]. Like the virus glycoproteins (gp85, gp37) [24], this polypeptide appears to have type specific properties. Analyses similar to the ones carried out above were extended to avian leukosis viruses. In contrast to the marked differences which have been reported for the size

231 [38-40] and subunit distribution of the 70 S RNA [38,41 ] of these viruses, the polypeptide patterns are largely indistinguishable. Such studies are presently being extended to the transformation defective viruses and spontaneous non-transforming derivatives of avian sarcoma viruses in an effort to determine if the changes which occur in the nucleic acids are reflected in any way in the properties of the structural proteins. Similar approaches are being carried out in other laboratories employing recombinants of avian sarcoma and leukosis viruses (Lai and Vogt, personal communication).

viii. COMPARATIVE PROPERTIES OF AVIAN AND MAMMALIAN ONCORNAV1RUS POLYPEPTIDES In spite of their separation on the evolutionary scale, avian and mammalian C-type oncornaviruses share many properties in common including morphology, biochemistry, mode of replication and oncogenic spectrum. As pointed out by others [42,43], these similarities suggest a common evolutionary origin. Some additional features of the major polypeptides of the avian and mammalian agents (see also Green et al. [31 ]) are presented below which further emphasize the close relationship which exists between these viruses. The properties described are also very useful in the identification of the respective polypeptides.

Avian plO, mammalian p12 Both stain "red" with Coomassie blue in contrast to the other proteins which stain "blue". They are similar in this regard to the major glycoprotein which also exhibits a reddish stain. Like the glycoproteins, the stain diffuses with prolonged storage in acetic acid. Polypeptides obtained from the guanidine column are associated with measurable carbohydrate (in preparation) but it is not yet certain whether the carbohydrate is covalently bound to the protein. Neither is found in the respective virus core but rather appears to represent surface components of the virus [44]. Because of their acidic nature, these can be easily separated from the other major polypeptides by electrophoresis in urea-containing gels at neutral or high pH. Avian p12, mammalian plO Both are strongly basic proteins which migrate rapidly in urea-acrylamide gels at low pH. They are found in the respective cores of the viruses and represent the polypeptide most closely associated with the RNA [44,45]. After staining with Coomassie blue, application of a strong light to the stained band reveals a type of "fluorescence" which is present to a much smaller extent in some of the other proteins. In this regard, they resemble histone proteins which behave in a similar manner (Mrozek, S., personal communication). The effect is probably due to an unusual binding of the dye by the proteins. These components also share other unusual properties; they are soluble in acetone and 0.5 M perchloric acid (unpublished observations).

232

Avian p15, mammalian p15 Material in this molecular weight range is associated with the cores of the viruses but in low and variable yields [44]. In this regard this observation may represent artifactual reassociation of this protein with the core. Amino acid analyses indicated that they contain a high percentage of hydrophobic residues possibly explaining why these proteins have a tendency to aggregate [54]. Recent work has shown that p15 is primarily associated with the virus outer membrane (in preparation). A vian p 19 This component is absent for the most part in mammalian oncornaviruses. However, careful analysis has shown that a small quantity of material of this molecular weight does indeed exist in at least some of the mammalian agents (Fleissner, E., and Nowinski, R., personal communication). In this regard, it is of interest that avian sarcoma viruses of subgroup C seem to possess the least amounts of this polypeptide. Of all the avian agents, it is the subgroup C sarcoma viruses which most readily infect and transform mammalian cells. Could these have been the progenitors of the mammalian viruses? Avian p27, mammalian p31 In each case this component represents the major polypeptide of the respective viruses. They are also major constituents of the virus core, most likely representing a portion of the core shell [44]. Serologically, these are among the stronger antigens of the viruses [4,6,8,31 ]. A feature of the mammalian p31 is that it contains multiple antigenicities including type, group and interspecies specific determinants (for review see August et al. [30]). Avian gp85, mammalian gp 71" These glycoproteins are constituents of the outer envelope of avian and mammalian oncornaviruses. Their functional roles relate to the virus host range, interference pattern, and the capacity to induce neutralizing antibody (for review see: Bolognesi [46] and Bauer [47]). In the case of the Friend leukemia virus, they also demonstrate hemagglutinating capacity [35,36]. Structurally they represent the surface prqiections of the virus [27,35,36,48,49]. They have recently been isolated, purified extensively and characterized serologically [27, 33, 34, 50, 51]. Like p31, mammalian gp 71 reveals multiple antigenic determinants (for review see August et al. [30]) and a similar situation exists for avian gp85 [27] (in preparation). Both glycoproteins are acidic in the sense that they do not penetrate the urea gels at low pH. The acidity is probably due both to the carbohydrate moieties (sialic acids) and amino acids [52] (unpublished). These components can be identified by PAS staining or by labeling with carbobydrate precursors. Table lI summarizes the localization of the various components of avian and mammalian oncornaviruses on the basis of presently available information. In * These have different molecular weights but for nomenclature purposes are designated as gp71.

233 TABLE 11 LOCALIZATION OF MAJOR ONCORNAVIRUS STRUCTURAL COMPONENTS Avian Major 19oly19e19tides

Glycoproteins

pl0 1912 pl 5 p19 1927 gp37 gp85

Virion surface Core interior (RNP) Virion surface** Virion interior Core surface Surface spike Surface knob

Murine 1910 1912 1915 -1931 gp45 g1977

Core interior (RNP) Virion surface Virion surface** Absent Core surface Virion surface Virion surface

* These are associated with carbohydrates. ** lhle, J. N., Hanna, M. G., Sch~ifer, W., Hunsmann, G., Bolognesi, D. P., and H~iper,G. Virology, in press. addition to the polypeptides listed above, murine leukemia viruses reveal an additional component in the low molecular weight region of the gel. In MuLV (Friend) this polypeptide migrates between p12 and p15. However, in the guanidine column it elute; after p15 (Sch~ifer et al., [53]). There are, therefore, four polypeptides with molecular weights lower than p30 in MuLV (Friend) which are analogous to the avian system (Table 1I). It is not certain however, that all MuLV contain a similar number of components since indications have been obtained that one of these is present in variable strains (unpublished; R. Nowinski, and J. lhle, personal communications). IX. CONCLUDING REMARKS This review has focused attention on several biochemical properties of the major structural components of C-type oncornaviruses. Such knowledge is necessary for the understanding of the origin and relationship these agents have to one another and for the detection and identification of unknown R N A tumor viruses. It is also important information for studies concerning the analysis of virus gene products in the host cell. In addition to the physicochemical properties of these proteins, a great deal is known about their immunological aspects. The latter, which also represents very specific identification markers, have been the subject of recent extensive reviews [46,47]. Together with the biochemical properties, the immunological features of these proteins indicate that a m o n g the various agents, the corresponding structural polypeptides seem to have rather similar properties. It is now important to determine if they have functional roles in processes related to virus replication, cell transformation and tumorigenesis. ACKNOWLEDGEMENTS The authors are grateful to Dr A. J. Langlois for large quantities of A M V and MuLV (Friend). This work was supported by contract NO1 CP 33308 from The Virus Cancer Program of the National Cancer Institute.

234 REFERENCES 1 Varmus, H. E., Weiss, R. A., Friis, R. R., Levinson, W. and Bishop, J. M. (1972) Proc. Natl. Acad. Sci. U.S. 69, 20-24 2 Baluda,, M. (1972) Proc. Natl. Acad. Sci. U.S. 69, 576-580 3 Hayward, W. S. and Hanafusa, H. (1973) J. Virol. 11, 157-161 4 Bauer, H. and Bolognesi, D. P. (1970) Virology 42, 1113-1126 5 Cheung, K., Smith, R. E., Stone, M. P. and Joklik, W. K. (1972) Virology 50, 851-864. 6 Oroszlan, S., Foreman, C., Kelloff, G. and Gilden, R. V. (1971) Virology 43, 665-674 7 Gilden, R. V. and Oroszlan, S. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1021-1025 8 Sch/ifer, W., Lange, J., Fischinger, P. J., Frank, H., Bolognesi, D. P. and Pister, L. (1972) Virology 47, 210-228 9 Strand, M. and August, J. T. (1974) J. Vitrol. 13, 171-180 10 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 11 Bretscher, M. S. (1971) Nat. New Biol. 231,229-232 12 Fleissner, E. (1971) J. Virol. 8, 778-785 13 Nowinski, R. C., Fleissner, E., Sarkar, N. and Aoki, T. (1972) J. Virol. 9, 359-366 14 Fish, W., Mann, K. and Tanford, C. (1969) J. Biol. Chem. 244, 4989-4994 15 Tung, J. and Knight, C. A. (1972) Anal. Biochem. 48, 153-163. 16 Tung, J. and Knight, C. A. (1972) Virology 48, 574-581 17 Bauer, H. and Sch~ifer, W. (1965) Z. Naturforsch. B 20, 815-817. 18 Allen, D. W. (1967) Biochim. Biophys. Acta 133, 180-183 19 Hung, P. H., Robinson, H. L. and Robinson, W. H. (1971) Virology 30, 251-266 20 Moscovici, C. and Vogt, P. K. (1969) Virology 35, 487-497 21 Hanafusa, H. (1965) Virology 25, 248-255. 22 Graf, T., Bauer, H., Gelderblom, H. and Bolognesi, D. P. (1971) Virology 43, 427-441 23 Graf, T. (1972) Virology 50, 567-578 24 Graf, T. (1973) Virology 54, 398-413 25 Graf, T. (1972) Z. Naturforsch. B 27, 223-226 26 Bolognesi, D. P. and Bauer, H. (1970) Virology 42, 1097-1112 27 Bolognesi, D. P., Bauer, H., Gelderblom, H. and Huper, G. (1972) Virology 47, 551-566 28 Green, R. W. and Bolognesi, D. P. (1974) Anal. Biochem. 57, 108-117 29 Panyim, S. and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346 30 August, J. T., Bolognesi, D. P., Fleissner, E., Gilden, R. V. and Nowinski, R. C. (1974) Virology 60, 595-600 31 Green, R. W., Bolognesi, D. P., Sch/ifer, W., Pister, L., Hunsmann, G. and de Noronha, F. (1973) Virology 56, 565-579 32 Duesberg, P. H., Martin, G. S. and Vogt, P. K. (1970) Virology 41,631-646 33 Strand, M. and August, T. (1973) J. Biol. Chem. 248, 5627-5633. 34 Moennig, V., Hunsmann, G. and Sch~ifer, W. (1973) Z. Naturforsch. C 28, 785-788 35 Witter, R., Frank, H., Moennig, V., Hunsmann, G., Lange, J. and Sch~ifer, W.(1973) Virology 54, 330-345 36 Witter, R., Hunsmann, G., Lange, .1. and Sch~fer, W. (1973) Virology 54, 346-358 37 Ishizaki, R., Luftig, R. and Bolognesi, D. P. (1973) .I. Virol. 12, 1579-1588 38 Duesberg, P. H. and Vogt, P. K. (1970) Proc. Natl. Acad. Sci. U.S. 67, 1673-1680 39 Bolognesi, D. P. and Graf, T. (1971) Virology 43, 214-222 40 Scheele, C. M. and Hanafusa, H. (1972) Virology 50, 753-764 41 Duesberg, P. H. and Vogt, P. K. (1973) Virology 54, 207-219 42 Oroszlan, S., S., Copeland, T., Summers, M. and Gilden, R. V. (1972) Biochem. Biophys. Res. Commun. 48, 1549-1555 43 Oroszlan, S., Copeland, T., Summers, M. and Gilden, R. V. (1972) Science 181,454-456 44 Bolognesi, D. P., Luftig, R. and Shaper, J. (1973) Virology 56, 549-564 45 Fleissner, E. and Tress, E. (1973) J. Virol. 12, 1612-1615 46 Bolognesi, D. P. (1974) Adv. Virus Res. 19, in press 47 Bauer, H. (1974) Adv. Cancer Res. 20, in press 48 Rifkin, D. B. and Compans, R. W. (1971) Virology 46, 485-489 49 Gelderblom, H., Bauer, H., Bolognesi, D. P. and Frank, H. (1972) Zbl. Bakt. Hyg., 1. Abt.

235 Orig. A 220, 79-90 Moennig, V., Frank, H., Hunsmann, G., Schneider, 1. and Sch~fer, W. (1974) Virology, in press Hunsmann, G., Moennig, V., Pister, L., Seifert, S. and Schafer, W. (1974) Virology, in press Krantz, M. J. and Lee, Y. C. (1974) Nature 248, 684-686 Sch/ifer, W., Hunsmann, G., Moennig, V., de Noronha, F., Bolognesi, D. P., Green, R. W. and Hiiper, G, (1974) Virology, in press 54 Herman, A. C., Green, R. W., Bolognesi, D. P. and Vanaman, T. C. Virology, in press 55 Bolognesi, D. P., Fshizaki, R., Htiper, G., Vanaman, T. C. and Smith, L. Virology, in press 50 51 52 53